RNA adapter ligation

Adapter concentration will vary depending on overall RNA yield, see table below:

Quantity of starting material	Adapter stock concentration	Adapter concentration in ligation reaction
100 – 250 ng	140 nM	10 nM
251 – 500 ng	350 nM	25 nM
501 – 2000 ng	700 nM	50 nM
2001 – 4000 ng	1400 nM	100 nM

For Puritz and Lotterhos 2017, we used 4000 ng starting RNA, but because of difficulties assessing and quantifying molluscan RNA, we chose to use a 700 nM working stock with a final reaction concentration of 50 nM.

This will be where we insert the custom adapters that are barcoded with RE sites

• Set up the adapter ligation reactions as follows:

Component	Volume
Beads with A-tailed DNA	15 ul
Adapter Ligation Master Mix	17.5 ul
–Adapters—	2.5 ul
–Total Volume–	–35 ul–

• Mix thoroughly by pipetting up and down several times to resuspend the beads.
• Incubate the plate/tube at 20 °C for 30 min.
• Proceed immediately to –1st Post-Ligation Cleanup–.